The N-terminal extension of the P. falciparum GBP130 signal peptide is irrelevant for signal sequence function

The malaria parasite P. falciparum exports a large number of proteins to its host cell, the mature human erythrocyte. Although the function of the majority of these proteins is not well understood, many exported proteins appear to play a role in modification of the erythrocyte following invasion. Protein export to the erythrocyte is a secretory process that begins with entry to the endoplasmic reticulum. For most exported proteins, this step is mediated by hydrophobic signal peptides found towards the N-terminal end of proteins. The signal peptides present on P. falciparum exported proteins often differ in length from those found in other systems, and generally contain a highly extended N-terminal region. Here we have investigated the function of these extended N-terminal regions, using the exported parasite protein GBP130 as a model. Surprisingly, several deletions of the extended N-terminal regions of the GBP130 signal peptide have no effect on the ability of the signal peptide to direct a fluorescent reporter to the secretory pathway. Addition of the same N-terminal extension to a canonical signal peptide does not affect transport of either soluble or membrane proteins to their correct respective subcellular localisations. Finally, we show that extended signal peptides are able to complement canonical signal peptides in driving protein traffic to the apicoplast of the parasite, and are also functional in a mammalian cell system. Our study is the first detailed analysis of an extended P. falciparum signal peptide and suggests that N-terminal extensions of exported Plasmodium falciparum proteins are not required for entry to the secretory system, and are likely to be involved in other, so far unknown, processes.     

Mutational analysis of Plasmodium falciparum dihydrofolate reductase: the role of aspartate 54 and phenylalanine 223 on catalytic activity and antifolate binding

The catalytic activity and ability to confer resistance to antifolates of Plasmodium falciparum dihydrofolate reductase (pfDHFR) through single and double mutations at Asp-54 and Phe-223 were investigated. A single Asp54Glu (D54E) mutation in the pfDHFR domain greatly decreased the catalytic activity of the enzyme and affected both the K(m) values for the substrate dihydrofolate and the K(i) values for pyrimethamine, cycloguanil and WR99210. The Phe223Ser (F223S) single mutant had unperturbed kinetics but had very poor affinity with the first two antifolates. The ability to confer high resistance to the antifolates of F223S enzyme was, however, abolished in the D54E+F223S double mutant enzyme. When D54E mutation was present together with the A16V+S108T double mutation, the effects on the K(m) values for the substrate dihydrofolate and the binding affinity of antifolates were much more pronounced. The severely impaired kinetics and poor activity observed in A16V+S108T+D54E enzyme could, however, be restored when F223S was introduced, while the binding affinities to the antifolates remained poor. The experimental findings can be explained with a model for substrate and inhibitor binding. Our data not only indicate the importance of Asp-54 of pfDHFR in catalysis and inhibitor binding, but also provide evidence that infer the potentially crucial function of the C-terminal portion of pfDHFR domain.     

Identification of a mitochondrial superoxide dismutase with an unusual targeting sequence in Plasmodium falciparum

The intraerythrocytic stages of Plasmodium falciparum are exposed to oxidative stress and require functional anti-oxidant systems to survive. In addition to the parasite's known iron-dependent superoxide dismutase PfSOD1, a second SOD gene (PfSOD2) interrupted by 8 introns was identified on chromosome 6. Molecular modelling shows that the structure of PfSOD2 is similar to other iron-dependent SODs and phylogenetic analysis suggests PfSOD1 and PfSOD2 are the result of an ancestral gene duplication. The deduced amino acid sequence of PfSOD2 is similar to PfSOD1 but has a long N-terminal extension. Immunofluorescence studies show that PfSOD1 is cytosolic, whereas the N-terminal extension of PfSOD2 targets a green fluorescent protein fusion into the parasite's mitochondrion. Both SOD genes are transcribed during the erythrocytic cycle with PfSOD1 mRNA levels up to 35-fold higher than those of PfSOD2. Northern blots demonstrated that the mRNA levels of both SOD genes are up-regulated upon exposure to oxidative stress.     

Immunogenicity of a non-repetitive sequence of Plasmodium falciparum circumsporozoite protein in man and mice

In the present work, the hypothesis that individuals naturally exposed to Plasmodium falciparum malaria infection in endemic areas produce antibodies directed against non-repetitive epitopes of the circumsporozoite protein was investigated. Using a synthetic peptide reproducing the non-repetitive group-conserved region I sequence, we have shown that specific anti-region I antibodies are detectable in sera from endemic countries. Of these sera, 87% also had antibodies against the immunodominant repetitive epitope (Asn-Ala-Asn-Pro, NANP) of P. falciparum. In order to study the immunogenicity of this non-repetitive epitope, a synthetic peptide consisting of both region I and three (NANP) repeats [RI-(NANP)3] was used to immunize inbred strains of mice. H-2b mice produced antibodies against both the repetitive and the non-repetitive epitope. These antibodies were specific for each epitope, recognized P. falciparum sporozoites in immunofluorescence, and inhibited sporozoite penetration into human liver cells in vitro. Non-H-2b mice were completely unresponsive. Lymph node cells from H-2b mice immunized with RI-(NANP)3 peptide proliferated in the presence of RI-(NANP)3 and of (NANP)4 peptide, but never in the presence of RI peptide alone. These findings demonstrate that in the configuration used (i) the non-repetitive epitope region I does not carry T-helper epitopes; (ii) the (NANP) repetitive epitope may act as a carrier for the immune response to region I in mice; and (iii) therefore, immune response to region I in man probably depends on the recognition of T-cell epitopes similar to those involved in the anti-NANP response: i.e. such a T epitope may be NANP itself in responding individuals or another, not yet recognized, sporozoite T-cell epitope.     

Parasite-derived mitogenic activity for human T cells in Plasmodium falciparum continuous cultures.

Supernatants from Plasmodium falciparum continuous cultures exhibited mitogenic activity against human blood lymphocytes from unsensitized donors. This effect, which was not observed with supernatants from control cultures grown in the absence of the parasites, was dependent upon (i) the concentration of supernatant added to the lymphocyte cultures and (ii) the parasite concentration in the P. falciparum continuous cultures. T cells were the predominant target cells of this mitogenic activity. We observed similar response in lymphocytes from malaria-sensitized individuals to P. falciparum continuous culture material. We also detected a mitogenic activity in parasite-infected erythrocytes from P. falciparum continuous cultures. P. falciparum continuous cultures may provide practical quantities of parasite-derived substances which, presumably, are able to manipulate the immune effector mechanisms of an infected host.     

Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum

The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.     

Monoclonal antibodies to a synthetic peptide corresponding to a repeated sequence in the Plasmodium falciparum antigen Pf155

Mouse monoclonal antibodies were prepared against a synthetic peptide (EENVEHDA) corresponding to a tandemly repeated sequence in the C-terminus of the Plasmodium falciparum antigen Pf155. One antibody (IgG1) producing hybridoma was studied in detail. The specificity of the antibody was determined by enzyme-linked immunosorbent assays using bovine serum albumin-conjugated or free peptides as solid phase antigens and various synthetic peptides for inhibition. The antibody reacted with Pf155 as detected by immunofluorescence and immunoblotting. It was also an efficient inhibitor of merozoite invasion in P. falciparum in vitro cultures indicating that it defines a biologically important epitope present on the native Pf155 molecule.     

Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.     

Evidence for prenylation-dependent targeting of a Ykt6 SNARE in Plasmodium falciparum

Ykt6 proteins are the most versatile fusogens in eukaryotic cells, and the only SNAREs that can be both prenylated and acylated at a C-terminal CAAX motif. Unlike yeast and mammalian cells where a single Ykt6 gene is expressed, the Plasmodium falciparum genome encodes two Ykt6 proteins. We have investigated the expression and prenylation of the Ykt6 orthologue, PfYkt6.1 in intra-erythrocytic stages of P. falciparum. PfYkt6.1 localized to the parasite Golgi and other unidentified cytoplasmic compartments, and was partly cytosolic (～50% in early trophozoites). The membrane-association of PfYkt6.1 was dependent on the presence of a conserved C-terminal CAAX motif (CCSIM). By expressing full-length and mutant proteins in Escherichia coli, we have shown that PfYkt6.1 indeed serves as substrate for prenylation by P. falciparum farnesyltransferases. Surprisingly, PfYkt6.1 could also be geranylgeranylated by parasite extracts independent of the C-terminal amino acid residue. Deletion of the CAAX motif inhibited both farnesylation and geranylgeranylation activities. Additionally, the PfYkt6.1 heptapeptide KQCCSIM, corresponding to the C-terminal CAAX sequence, inhibited the parasite farnesyltransferase activity with an IC(50) of 1 μM. Our findings underscore the importance of CAAX motif-derived peptidomimetics for antimalarial drug development.     

Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum

Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 microg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.     

A dual expression system for the generation, analysis and purification of antibodies to a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA

A novel dual expression system for the generation and analysis of immune responses to recombinant protein is described. The two expression systems are based on the IgG-binding domains (ZZ) of staphylococcal protein A (SpA) and the human serum albumin (HSA) binding domains (BB) of streptococcal protein G, respectively. Products of fusions with the ZZ region are used to generate an immune response against the recombinant peptide and the corresponding peptide fused to the BB region is used for analysis and purification of the specific antibodies. The protein A and protein G expression systems were used to produce fusion proteins with the repeated C terminal octapeptide subunit EENVEHDA of the Plasmodium falciparum merozoite derived protein Pf155/RESA. Rabbits were immunized with the protein A-derived fusion protein (designated ZZ-M1) and the antibody response was analyzed using the protein G-derived fusion protein (designated BB-M1). The rabbit antisera reacted with BB-M1 in both ELISA and immunoblotting. In addition, BB-M1 proved to be an efficient ligand for affinity purification of antibodies specific for the malaria peptide. Furthermore, the rabbit antisera reacted with Pf155/RESA both in merozoite extracts and when deposited in the membrane of parasite infected erythrocytes.     

Food vacuole plasmepsins are processed at a conserved site by an acidic convertase activity in Plasmodium falciparum

Intraerythrocytic Plasmodium falciparum digests vast amounts of hemoglobin within an acidic food vacuole (FV). Four homologous aspartic proteases participate in hemoglobin degradation within the FV. Plasmepsin (PM) I and II are thought to initiate degradation of the native hemoglobin molecule. PM IV and histo-aspartic protease (HAP) act on denatured globin further downstream in the pathway. PM I and II have been shown to be synthesized as zymogens and activated by proteolytic removal of a propiece. In this study, we have determined that the proteolytic processing of FV plasmepsins occurs immediately after a conserved Leu-Gly dipeptidyl motif with uniform kinetics and pH and inhibitor sensitivities. We have developed a cell-free in vitro processing assay that generates correctly processed plasmepsins. Our data suggest that proplasmepsin processing is not autocatalytic, but rather is mediated by a separate processing enzyme. This convertase requires acidic conditions and is blocked only by the calpain inhibitors, suggesting that it may be an atypical calpain-like protease.     

Plasmodium falciparum: a comparative analysis of the genetic diversity in malaria-mesoendemic areas of Brazil and Madagascar

For a better definition of the polymorphic features of Plasmodium falciparum parasite populations, the polymerase chain reaction (PCR) typing technique was used to investigate the genetic diversity and complexity of parasites harbored by acute P. falciparum carriers from three yet unexplored malaria-mesoendemic areas with different transmission levels: two localities in northwestern Brazil (Ariquemes and Porto Velho) and a village in Madagascar (Ankazobe). A total of 89 DNA samples were analyzed by amplification of polymorphic domains from genes encoding merozoite surface antigens 1 and 2 (MSP-1, MSP-2) and thrombospondin-related anonymous protein (TRAP) and by hybridization with allelic-family-specific probes or random-fragment-length polymorphism (RFLP). In all three localities, extensive polymorphism was observed for each marker, but the MSP-2 central repeat was the most diverse one. Similar levels of genetic diversity, allelic frequency, and infection complexity were observed in the two Brazilian localities, although the isolates had been sampled at 2-year intervals, suggesting the stability of the infecting parasite populations presenting in these regions of the Brazilian Amazon. Unexpectedly, although the entomologic inoculation rate was at least 3 times lower in Ankazobe than in the Brazilian areas, Malagasi samples appeared more complex than the Brazilian ones. The implications of these data with regard to parasite population-dynamics studies are discussed. Received: 5 September 1999 / Accepted: 1 October 1999     

Mutational analysis of p53 and RB2/p130 genes in Malaysian nasopharyngeal carcinoma samples: a preliminary report

This study reports the results of mutation detection of tumour suppressor genes, p53 and RB2/p130 genes in Malaysian nasopharyngeal carcinoma (NPC) studied by PCR-CSGE analysis and direct DNA sequencing method. Frequent sites of mutation in both genes (exons 5-8 of p53 and exons 19-21 of RB2/p130) were examined. Thirty-six NPC blood samples and three NPC cell lines were investigated for the presence of mutations. No mutation of p53 and RB2/p130 genes was identified in any of the blood samples. Nonetheless, there was an identical G-->4 C nucleotide change at codon 280 of p53 gene in all the cell lines. A larger study that includes biopsy tissues should be carried out to provide a more in-depth look into the pathogenesis of NPC in Malaysia.